Fig 2: OPCs thrive and morphologically develop in the contusion injury site. (A) Mature oligodendrocyte marker APC and transplanted GFP-OPCs at 10 days and 6 weeks time points, counterstained with DAPI. (B) GFP-OPCs increase in morphological complexity over 6 weeks in association with SOX10+ cells in the contusion injury microenvironment. Ten days and 6 weeks time points shown with DAPI. (C) GFP-OPCs survive and integrate within the GFAP+ injury milieu by 6 weeks. (D) GFP-OPCs arborize in the contusion injury microenvironment. O4 staining shown with DAPI. (E,F) Analysis of GFP-OPC and GFAP signal overlap at 6 weeks. GFAP and GFP signal is distinct with regions of overlap along STEM121 neural processes. Two examples are provided with zoom images. Asterisks depict GFP-OPC (green), GFAP (magenta), STEM121 (red), and signal overlap (white). R (rostral), C (caudal). Individual scale bars are provided.

Fig 3: Continued survival of SMN-OPC cell suspensions at 6 weeks post-transplantation. (A) Low power image of the graft at 6 weeks shown with STEM121/GFP-OPC/GFAP to depict host glial cells. (B) Low power image of the graft at 6 weeks shown with STEM121/GFP-OPC and TUJ1 to validate retention of the neuronal phenotype. White box denotes high magnification field shown in (C), imaged caudal to the lesion epicenter. (C) High magnification merged image and individual channels (STEM121/GFP-OPC/TUJ1/GFAP) at 6 weeks post-transplantation. (D) STEM121 immunostain with mature neuronal marker NeuN co-localization. (E,F) Cavalieri volumetric analysis of the residual lesion and grafts. Color matching corresponds to the same animal. Shown are residual lesion vs. graft volume for individual spinal cords at 6 weeks (mm3) (E) and mean ± s.e.m (F). N = 3 animals were sacrificed at 6 weeks post-transplantation. R (rostral), C (caudal). Individual scale bars are provided.

Fig 4: Aβ1–42 elevated pre- and postsynaptic marker inside astrocytes. Anaesthetics restore Aβ1–42-induced elevation of astrocyte-dependent synaptic engulfment in hippocampal slices. (A,E) the red, green and yellow colours represent the astrocyte (GFAP, red), presynaptic marker (synaptophysin, green) and merge of GFAP and synaptophysin (yellow), respectively (top panels). The lower panels in (A,E) represent the same cell but rendered into 3D using IMARIS software (Bitplane). The raw image data (apotome and respective 3D IMARIS reconstructions) for (A,E) are seen Supplementary Figures S1A and S2A, respectively). (B,F) the results show an increase of synaptophysin stained material inside the astrocyte when treated with Aβ1–42 (% of engulfment, Mann–Whitney U test, * p = 0.0194). (C,G) the red, green and yellow colours represent the astrocyte (GFAP, red), postsynaptic marker (PSD95, green) and merge of GFAP and PSD95 (yellow), respectively. The lower panels in (C,G) represent the same cell but rendered into 3D using IMARIS software. The raw image data (apotome and respective 3D IMARIS reconstructions) for (C,G) are seen Supplementary Figures S1B and S2B, respectively. (D,H) Aβ1–42 elevated postsynaptic marker inside astrocytes (% of engulfment, Mann–Whitney U test, * p = 0.0078). (B,D,F,H) the dot plots showed that the combined application with either Iso or Xe reversed elevation of synaptic markers inside astrocytes (B, F, % of engulfment, Mann–Whitney U test/t-test, * p = 0.0241/0.0292. (D,H), % of engulfment, Mann–Whitney U test, * p = 0.0004/0.0002). Scale bar = 5 μm. Error bars in all graphs indicate the means +/− SDs. Every data point in the scatter blot represents one animal (n = 4–10). The images are from the brain slices of C57BL/6 mice, The % of engulfment of either pre- (synaptophysin) or post- (PSD95) synaptic material in (B,D,F,H) within astrocytes were assessed by employing the methods of Schafer et al., to our 3D IMARIS reconstruction images (see Methods; [38,39]).

Fig 5: Dagla is deleted in the progenitor-containing subgranular zone (SGZ) in GLAST-CreERT2-Dagla KO but not in Syn-Dagla KO mice. (A) Representative images of an RNAscope in situ hybridisation assay detecting transcripts of Dagla (white) in the DG in the hippocampus of Dagla fl/fl control, Syn-Dagla KO and GLAST-CreERT2-Dagla KO mice. Dagla is deleted in the neuronal granular cell layer in Syn-Dagla and in the progenitor-containing subgranular zone (SGZ) in GLAST-CreERT2-Dagla KO mice (scale bar 50 µm). The magnified insets (scale bar 10 µm) show SGZ with white arrows indicating Dagla expressing cells in Syn-Dagla KO and white arrowheads Dagla-negative cells in GLAST-CreERT2-Dagla KO mice in the NSC layer (n = 3–4 animals per group). (B) Immunostaining detecting DAGLa (magenta) co-localization with GFAP (yellow) and NeuN (green). The magnified insets show examples of GFAP-positive cells with NSPC morphology delineated with white. Progenitors express DAGLa in Dagla fl/fl control and Syn-Dagla KO SGZ and but not in GLAST-CreERT2-Dagla KO. NeuN positive neurons in granule cell layer express DAGLa in control and GLAST-CreERT2-Dagla KO sections, but not in Syn-Dagla KO (scale bar 10 µm).